Radiocarbon Flux Measurements Reveal Mechanistic Insight into Heat-Stress Induction of Nicotine Biosynthesis in Nicotiana attenuata.

The effect of high-temperature (HT) stress on nicotine biosynthesis in Nicotiana attenuata was examined. Nicotine content was measured in mature leaves, young sink leaves, and in roots from well-watered plants grown at 25 °C as controls and from plants exposed to 38 °C and 43 °C temperatures applied for 24, 48, 72, and 96 h duration. At 38 °C, all leaf nicotine levels were significantly less than control plants for up to 72 h exposure but rose sharply thereafter to levels significantly greater than controls with 96 h exposure. In contrast, plants exposed to 43 °C never exhibited a reduction in leaf nicotine content and showed an increase in content with just 48 h exposure. Using radioactive 11CO2 and 13NO3-, we found that HT stress reduced both CO2 fixation and nitrate uptake. Furthermore, radiocarbon flux analysis revealed that 'new' carbon partitioning (as 11C) into the 11C-radiolabeled amino acid (AA) pool was significantly reduced with HT stress as were yields of [11C]-aspartic acid, an important AA in nicotine biosynthesis, and its beta-amido counterpart [11C]-asparagine. In contrast, [12C]-aspartic acid levels appeared unaffected at 38 °C but were elevated at 43 °C relative to controls. [12C]-Asparagine levels were noted to be elevated at both stress temperatures. Since HT reductions in carbon input and nitrogen uptake were noted to impede de novo AA biosynthesis, protein degradation at HT was examined as a source of AAs. Here, leaf total soluble protein (TSP) content was reduced 39% with long exposures to both stress temperatures. However, Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) which was 41% TSP appeared unaffected. Altogether, these results support the theory that plant proteins other than Rubisco degrade at elevated temperatures freeing up essential AAs in support of nicotine biosynthesis.

Treatments with Liquid Smoke and Certain Chemical Constituents Prevalent in Smoke Reduce Phloem Vascular Sectoriality in the Sunflower with Improvement to Growth.

Many higher plants possess a physiological organization that is based upon the carbon economy of their parts. While photosynthates are partitioned according to the relative strength of the plant's sink tissues, in many species there is also a very close relationship between partitioning, phyllotaxy and vascular connectivity giving rise to sectorial patterns of allocation. Here, we examined the influence of smoke and certain chemical constituents prevalent in smoke including, catechol, resorcinol and hydroquinone on phloem vascular sectoriality in common sunflower (Helianthis annuus L.), as a model plant for sectoriality. By administering radioactive carbon-11 to a single source leaf as 11CO2, 11C-photosynthate allocation patterns were examined using autoradiography. A 1:200 aqueous dilution of liquid smoke treated soil caused 2.6-fold and 2.5-fold reductions in phloem sectoriality in sink leaves and roots, respectively. Treatment with catechol (1,2-d ihydroxybenzene) or resorcinol (1,3-dihydroxybenzene), polyphenolic constituents that are prevalent in smoke, caused similar reductions in phloem sectoriality in the same targeted sink tissues. However, treatment with hydroquinone (1,4-dihydroxybenzene) had no effect. Finally, the longer-term effects of smoke exposure on plant growth and performance were examined using outdoor potted plants grown over the 2022 season. Plants exposed to liquid smoke treatments of the soil on a weekly basis had larger thicker leaves possessing 35% greater lignin content than untreated control plants. They also had thicker stems although the lignin content was the same as controls. Additionally, plants exposed to treatment produced twice the number of flowers with no difference in their disk floret diameters as untreated controls. Altogether, loss of phloem sectoriality from exposure to liquid smoke in the sunflower model benefited plant performance.

Defense Priming in Nicotiana tabacum Accelerates and Amplifies 'New' C/N Fluxes in Key Amino Acid Biosynthetic Pathways.

In the struggle to survive herbivory by leaf-feeding insects, plants employ multiple strategies to defend themselves. One mechanism by which plants increase resistance is by intensifying their responsiveness in the production of certain defense agents to create a rapid response. Known as defense priming, this action can accelerate and amplify responses of metabolic pathways, providing plants with long-lasting resistance, especially when faced with waves of attack. In the work presented, short-lived radiotracers of carbon administered as 11CO2 and nitrogen administered as 13NH3 were applied in Nicotiana tabacum, to examine the temporal changes in 'new' C/N utilization in the biosynthesis of key amino acids (AAs). Responses were induced by using topical application of the defense hormone jasmonic acid (JA). After a single treatment, metabolic partitioning of recently fixed carbon (designated 'new' carbon and reflected as 11C) increased through the shikimate pathway, giving rise to tyrosine, phenylalanine and tryptophan. Amplification in 'new' carbon fluxes preceded changes in the endogenous (12C) pools of these AAs. Testing after serial JA treatments revealed that fluxes of 'new' carbon were accelerated, amplified and sustained over time at this higher rate, suggesting a priming effect. Similar results were observed with recently assimilated nitrogen (designated 'new' nitrogen reflected as 13N) with its partitioning into serine, glycine and glutamine, which play important roles supporting the shikimate pathway and downstream secondary metabolism. Finally, X-ray fluorescence imaging revealed that levels of the element Mn, an important co-factor for enzyme regulation in the shikimate pathway, increased within JA treated tissues, suggesting a link between plant metal ion regulation and C/N metabolic priming.

Inhibition of trehalose breakdown increases new carbon partitioning into cellulosic biomass in Nicotiana tabacum.

Validamycin A was used to inhibit in vivo trehalase activity in tobacco enabling the study of subsequent changes in new C partitioning into cellulosic biomass and lignin precursors. After 12-h exposure to treatment, plants were pulse labeled using radioactive (11)CO(2), and the partitioning of isotope was traced into [(11)C]cellulose and [(11)C]hemicellulose, as well as into [(11)C]phenylalanine, the precursor for lignin. Over this time course of treatment, new carbon partitioning into hemicellulose and cellulose was increased, while new carbon partitioning into phenylalanine was decreased. This trend was accompanied by a decrease in phenylalanine ammonia-lyase activity. After 4d of exposure to validamycin A, we also measured leaf protein content and key C and N metabolite pools. Extended treatment increased foliar cellulose and starch content, decreased sucrose, and total amino acid and nitrate content, and had no effect on total protein.

Partitioning of new carbon as ¹¹C in Nicotiana tabacum reveals insight into methyl jasmonate induced changes in metabolism.

We examined the timeline by which methyl jasmonate (MeJA) reprograms new carbon partitioning into key metabolite pools. The radioactive isotope ¹¹C (t(¹/2) 20.4 min), administered to intact leaves of Nicotiana tabacum L. (cv Samsun) as ¹¹CO(2) gas enabled us to measure changes in new carbon partitioning into soluble sugar and amino acid pools of [¹¹C]photosynthate. A 500 µM MeJA treatment resulted in a decrease in the [¹¹C]soluble sugar pool and an increase in the [¹¹C]amino acid pool after 4 h. This pattern was more pronounced 15 h after treatment. We also examined the timeline for ¹¹C-partitioning into aromatic amino acid metabolites of the shikimate pathway. [¹¹C]Tyrosine, [C¹¹C]phenylalanine and [¹¹C]tryptophan were elevated 1.5-fold, 12-fold and 12-fold, respectively, relative to controls, 4 h after MeJA treatment, while endogeneous pools were unchanged. This suggests that only new carbon is utilized during early stages of defense induction. By 15 h, [C¹¹C]tyrosine and [¹¹C]phenylalanine returned to baseline while [¹¹C]tryptophan was elevated 30-fold, suggesting that MeJA exerts selective control over the shikimate pathway. Finally, we measured trans-cinnamic acid levels as a gauge of downstream phenolic metabolism. Levels were unchanged 4 h after MeJA treatment relative to controls, but were increased 2-fold by 15 h, indicating a lag in response of secondary metabolism.

Use of gaseous 13NH3 administered to intact leaves of Nicotiana tabacum to study changes in nitrogen utilization during defence induction.

Nitrogen-13 (t(1/2) 9.97 m), a radioactive isotope of nitrogen, offers unique opportunities to explore plant nitrogen utilization over short time periods. Here we describe a method for administering (13)N as gaseous (13)NH(3) to intact leaves of Nicotiana tabacum L. (cv Samsun), and measuring the labelled amino acids using radio high-performance liquid chromatography (HPLC) on tissue extract. We used this method to study the effects of defence induction on plant nitrogen utilization by applying treatments of methyl jasmonate (MeJA), a potent defence elicitor. MeJA caused a significant increase relative to controls in key [(13)N]amino acids, including serine, glycine and alanine by 4 h post-treatment, yet had no effect on (13)NH(3) incorporation, a process that is primarily under the control of the glutamine synthatase/glutamate synthase pathway (GS/GOGAT) in cellular photorespiration. We suggest that the reconfiguration of nitrogen metabolism may reflect induction of non-photorespiratory sources of nitrogen to better serve the plant's defences.

Synthesis and PET studies of [(11)C-cyano]letrozole (Femara), an aromatase inhibitor drug.

INTRODUCTION: Aromatase, a member of the cytochrome P450 family, converts androgens such as androstenedione and testosterone into estrone and estradiol, respectively. Letrozole (1-[bis-(4-cyanophenyl)methyl]-1H-1,2,4-triazole; Femara) is a high-affinity aromatase inhibitor (K(i)=11.5 nM) that has Food and Drug Administration approval for breast cancer treatment. Here we report the synthesis of carbon-11-labeled letrozole and its assessment as a radiotracer for brain aromatase in the baboon. METHODS: Letrozole and its precursor (4-[(4-bromophenyl)-1H-1,2,4-triazol-1-ylmethyl]benzonitrile) were prepared in a two-step synthesis from 4-cyanobenzyl bromide and 4-bromobenzyl bromide, respectively. The [(11)C]cyano group was introduced via tetrakis(triphenylphosphine)palladium(0)-catalyzed coupling of [(11)C]cyanide with the bromo precursor. Positron emission tomography (PET) studies in the baboon brain were carried out to assess regional distribution and kinetics, reproducibility of repeated measures and saturability. Log D, the free fraction of letrozole in plasma and the [(11)C-cyano]letrozole fraction in arterial plasma were also measured. RESULTS: [(11)C-cyano]Letrozole was synthesized in 60 min with a radiochemical yield of 79-80%, with a radiochemical purity greater than 98% and a specific activity of 4.16+/-2.21 Ci/mumol at the end of bombardment (n=4). PET studies in the baboon revealed initial rapid and high uptake and initial rapid clearance, followed by slow clearance of carbon-11 from the brain, with no difference between brain regions. Brain kinetics was not affected by coinjection of unlabeled letrozole (0.1 mg/kg). The free fraction of letrozole in plasma was 48.9%, and log D was 1.84. CONCLUSION: [(11)C-cyano]Letrozole is readily synthesized via a palladium-catalyzed coupling reaction with [(11)C]cyanide. Although it is unsuitable as a PET radiotracer for brain aromatase, as revealed by the absence of regional specificity and saturability in brain regions such as amygdala, which are known to contain aromatase, it may be useful in measuring letrozole distribution and pharmacokinetics in the brain and peripheral organs.

11C-imaging: methyl jasmonate moves in both phloem and xylem, promotes transport of jasmonate, and of photoassimilate even after proton transport is decoupled.

The long-distance transport and actions of the phytohormone methyl jasmonate (MeJA) were investigated by using the short-lived positron-emitting isotope 11C to label both MeJA and photoassimilate, and compare their transport properties in the same tobacco plants (Nicotiana tabacum L.). There was strong evidence that MeJA moves in both phloem and xylem pathways, because MeJA was exported from the labeled region of a mature leaf in the direction of phloem flow, but it also moved into other parts of the same leaf and other mature leaves against the direction of phloem flow. This suggests that MeJA enters the phloem and moves in sieve tube sap along with photoassimilate, but that vigorous exchange between phloem and xylem allows movement in xylem to regions which are sources of photoassimilate. This exchange may be enhanced by the volatility of MeJA, which moved readily between non-orthostichous vascular pathways, unlike reports for jasmonic acid (which is not volatile). The phloem loading of MeJA was found to be inhibited by parachloromercuribenzenesulfonic acid (PCMBS) (a thiol reagent known to inhibit membrane transporters), and by protonophores carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP) suggesting proton co-transport. MeJA was found to promote both its own transport and that of recent photoassimilate within 60 min. Furthermore, we found that MeJA can counter the inhibitory effect of the uncoupling agent, CCCP, on sugar transport, suggesting that MeJA affects the plasma membrane proton gradient. We also found that MeJA's action may extend to the sucrose transporter, since MeJA countered the inhibitory effects of the sulfhydryl reagent, PCMBS, on the transport of photoassimilate.

Stimulating natural defenses in poplar clones (OP-367) increases plant metabolism of carbon tetrachloride.

Groundwater contamination by carbon tetrachloride (CCl4) presents a health risk as a potential carcinogen and pollutant that is capable of depleting the ozone layer. Although use of poplar trees in a phytoremediation capacity has proven to be cost effective for cleaning contaminated sites, minimizing leaf emission of volatile contaminants remains a pressing issue. We hypothesized that recently fixed carbon plays a key role in CCl4 metabolism in planta yielding nonvolatile trichloroacetic acid (TCA) and that the extent of this metabolism can be altered by heightening plant defenses. Labeling intact leaves with (11)CO2 (t 1/2 20.4 m) can test this hypothesis, because the extremely short half-life of the tracer reflects only those processes involving recently fixed carbon. Using radio-HPLC analysis, we observed [(11)C]TCA from leaf extract from poplar clones (OP-367) whose roots were exposed to a saturated solution of CCl4 (520 ppm). Autoradiography of [(11)C]photosynthate showed increased leaf export and partitioning to the apex within 24 h of CCl4 exposure, suggesting that changes in plant metabolism and partitioning of recently fixed carbon occur rapidly. Additionally, leaf CCl4 emissions were highest in the morning, when carbon pools are low, suggesting a link between contaminant metabolism and leaf carbon utilization. Further, treatment with methyljasmonate, a plant hormone implicated in defense signal transduction, reduced leaf CCl4 emissions two-fold due to the increased formation of TCA.